This project's goal is to understand how eukaryotic cells direct specific proteins along a pathway leading to secretion of those proteins. Recent advances suggest that the precursors of secreted proteins themselves direct those proteins along the secretory pathway and that proteins cross the endoplasmic reticulum while they are still being synthesized. Precisely how the precursors help transport proteins across microsomal membranes and why ongoing protein synthesis is required are not understood. To answer these questions, mutations are being introduced into the gene encoding preproPTH, the precursor of a secreted protein. The phenotypic consequences of these alterations are then determined by expressing the altered genes in clonal rat pituitary cells. The precise biochemical lesions are characterized further in cell-free extracts. A cell-free transcription-translation system is used that allows the transcription and translation of cloned DNA encoding secreted proteins and the concomitant transport and processing of the secreted proteins across microsomal membranes. Enzymatic methods and synthetic oligonucleotides are being used to generate mutations in the sequences thought to be involved in the secretion of proteins. We want to determine the functions 1) of positively charged residues near the NH2-termini of these precursors of secreted proteins, 2) of hydrophobic sequences near the NH2-termini of these precursors, 3) of residues that direct the cleavage of peptides from the precursors as they cross the microsomal membrane, and 4) of sequences just distal to the cleavage site. These sequences may be important in the reversible binding of polysomes to signal recognition particles, in the insertion of the protein into the microsomal membrane, or in the transport of the protein completely through the membrane. Further, the ribosome might be important for interactions with signal recognition particles, might serve as part of an engine driving proteins across membranes, or might direct the nascent protein to specific membrane channels. The experiments proposed here will allow us to assign specific functions to the precursor sequences and to the ribosome and will help define intermediate steps in the secretory pathway. This project thus uses a genetic approach to study a fundamental question in cell biology: How do cells direct proteins to specific cellular compartments to generate the cell's highly ordered structure?